Development of gene therapy with a cyclic adenosine monophosphate response element decoy oligodeoxynucleotide to prevent vascular intimal hyperplasia

Abstract

ObjectiveIntimal hyperplasia (IH) is the main cause of therapeutic failure after vascular and endovascular surgery. However, there is currently no targeted therapy for the treatment of IH. We recently reported that the inhibition of cyclic adenosine monophosphate response element (CRE) binding protein (CREB) activation is important in vein graft IH. We focused on a decoy oligodeoxynucleotide (ODN) therapeutic strategy for suppressing IH as a clinical application. The objective of this study was to confirm the therapeutic effect of a CRE decoy ODN in an animal model as a novel therapy for preventing intimal hyperplasia as the first step of the preclinical study of our strategy. MethodsWe designed two phosphorothioate CREs and two scramble decoy ODNs and screened them using a CREB transcription assay to check their ability to bind to a CRE sequence. We chose a CRE decoy ODN with high first-binding ability and transfected it into vascular smooth muscle cells (VSMCs) in vitro. Proliferation and migration were assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays and modified Boyden chamber assays. We examined CRE activity using a luciferase reporter gene assay. We assessed the expression of messenger RNAs by quantitative real-time polymerase chain reaction. In a wire-injury mouse model (C57BL6, n = 6), CRE decoy ODN was transfected into the injured vessel wall using an ultrasound-sonoporation method in vivo. Mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) and four and a half LIM domains 5 (FHL5) expression of pregrafting vein remnants were assessed by immunohistologic analyses. ResultsCompared with scramble decoy ODN, the selected CRE decoy ODN could significantly decrease CRE activity (mean ± standard error of the mean: 0.20 ± 0.03 vs 1.00 ± 0.16, n = 6; P < .05) as shown by a luciferase reporter gene assay, VSMC proliferation (0.73 ± 0.04 vs 0.89 ± 0.02, n = 6; P < .05) and migration (96.4 ± 6.1 vs 311.4 ± 19.1 migrated VSMCs/well, n = 6; P < .05) after 24-hour transfection. The CRE decoy ODN significantly suppressed the formation of IH at injured vessel walls in an animal model, as analyzed by pathologic staining (0.20 ± 0.02 vs 0.56 ± 0.08, area of the intima/area of the artery vs the control after 21 days’ transfection, n = 6; P < .05). Furthermore, MAPKAPK3 and FHL5, which are CREB activators, were significantly expressed in pregrafting vein remnants in diabetes mellitus patients. ConclusionsCREB-CRE signaling is an important mechanism of IH formation, and CRE decoy therapy can help preventing IH. This study is the first part of the preclinical study of our strategy.